Peptides with antioxidant and antimicrobial properties

ABSTRACT

Methods of treating conditions associated with lipid oxidation or microbial proliferation include the step of administering a composition comprising a pharmacologically effective amount of an antioxidant or antimicrobial lung surfactant protein compound. Peptides derived from lung surfactant protein compounds possess lipid oxidation inhibiting and/or antimicrobial properties.

RELATED APPLICATION

The present application claims priority under 35 U.S.C. § 119 of U.S.Provisional Application Ser. No. 60/215,313 filed Jun. 30, 2000, theentire disclosure which is herein incorporated by reference.

This invention was made with government support under Grant No. HL-61612awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods for reducing orpreventing the oxidation of lipids or other molecules, and to methodsfor inhibiting or reducing microbial proliferation. Specifically, thepresent invention is directed to compositions and methods for treatingoxidative lung injury and other oxidative disorders, and to methods forpreventing oxidation of foods, cosmetics and medications. Additionally,the present invention is directed to compositions and methods fortreating lung infection and other disorders caused by microbialinfection.

The lung is made up of a series of branching conducting airways thatterminate in grape-like clusters of delicate gas exchanging airspacescalled pulmonary alveoli. Maintenance of alveolar patency at endexpiration requires pulmonary surfactant, a mixture of saturated andunsaturated phospholipids and proteins that lines the epithelial surfaceand reduces surface tension. Surfactant is presented to the oxidizingeffects of atmospheric oxygen and inhaled toxicants over a tenniscourt-sized interface with the environment. The pathophysiologicalconsequences of surfactant oxidation in humans and experimental animalsexposed to hyperoxia include airspace collapse, reduced lung complianceand impaired gas exchange.

Air breathing is made possible through the surface tension loweringproperties of lung surfactant, an oily film located at the boundarybetween the aqueous pulmonary epithelial lining fluid (ELF) and the airin the lumen of the alveoli, the gas exchanging distal airspaces of thelung. By weight, surfactant is composed of approximately 50% saturatedphospholipids, 40% unsaturated phospholipids and 10% protein, includinghydrophilic surfactant proteins A (SP-A) and D (SP-D), and thehydrophobic surfactant proteins B (SP-B) and C (SP-C). After secretioninto the ELF, the components of surfactant form membranes at theair-liquid interface which spread readily and compress poorly duringcyclical respiratory expansion and contraction.

These properties of surfactant result in enhanced lung compliance duringinspiration, which reduces the work of breathing, and very low alveolarsurface tension at end expiration, which helps to maintain airspacepatency. Exposure of surfactant to ambient oxygen and potentenvironmental oxidants such as ozone can result in peroxidation ofunsaturated phospholipids, surfactant inactivation, airspace collapseand impaired gas exchange. Antioxidant protection of surfactantphospholipids in the ELF has classically been attributed to lowmolecular mass components urate, ascorbate, and reduced glutathione, andto proteinaceous antioxidants albumin, superoxide dismutase andcatalase.

Moreover, after secretion by alveolar type II cells and nonciliatedbronchiolar cells into the alveolar lining layer (ALL), the componentsof surfactant form phospholipid enriched membranes at the air-liquidinterface. The surfactant lining has critical surface tension loweringproperties which reduce the work of breathing and help to maintainairspace patency. However, the surfactant layer also places ahydrophobic barrier between the inhaled organism and the antimicrobialdefenses of the pulmonary epithelium and ALL. In the absence of anyspecialized defense mechanisms positioned in and around the surfactantmembrane, the organism could theoretically proliferate in amicroenvironment free from the threat of phagocytic cells, specificantibodies, or innate immune antimicrobial peptides. Without being boundby theory, it is believed that the hydrophilic protein components of thesurfactant lining layer, SP-A and SP-D, have potent macrophageindependent antimicrobial properties.

The oxidative modifications of surfactant lipids and low densitylipoproteins (LDL) and/or microbial proliferation represent key eventsin the pathogenesis of tissue injury. Thus there is a need for usefultherapeutical application of compounds with antioxidant propertiesand/or antimicrobial properties in the treatment of the inflammatory andhyperoxic lung disease, atherosclerosis, oxidative injury to the skin,and/or lung infection or injury. There is also a need to preventspoilage, off flavors and off colors due to oxidation and/or microbialproliferation in foods, cosmetics and medications.

SUMMARY OF THE INVENTION

An object of the invention is to provide methods for inhibiting theoxidation of lipids, proteins and other compounds. An additional objectis to provide methods for treating conditions associated with microbialcontamination, colonization or infection. Another object of the presentinvention is to provide methods and compositions for treating a mammalhaving a condition associated with oxidative tissue injury, such asoxidative lung injury, aging of the skin or atherosclerosis. Similarly,another object of the present invention is to provide methods forinhibiting a macrophage-independent microbial proliferation in mammalswith lung infection or injury, or systemic or local infections. Yetanother object of the invention is to provide compositions and methodsfor the treatment of acute lung injury, including adult respiratorydistress syndrome and hyperoxic lung injury.

One aspect of the invention is the prevention of oxidation in food,pharmaceutical compositions, cosmetic preparations and dermatologicalpreparations. Similarly, another aspect of the invention is theprevention of microbial proliferation in food, pharmaceuticalcompositions, cosmetic preparations and dermatological preparations.

Another object of the invention is to provide pharmacologicalcompositions comprising antioxidant or antimicrobial lung surfactantprotein compounds. One method of inhibiting lipid oxidation or microbialproliferation is by intratracheal, dermal or oral administration of anantioxidant or antimicrobial lung surfactant protein compound to amammal, preferably a human.

In one embodiment, pharmacological compositions may comprise anantioxidant or antimicrobial lung surfactant protein compound and,optionally, pharmaceutically acceptable carriers, fillers or excipients.In another embodiment, a method of treating a mammal, preferably ahuman, comprises administering to the mammal a pharmacologicalcomposition comprising an antioxidant or antimicrobial lung surfactantprotein compound and, optionally, pharmaceutically acceptable carriers,fillers or excipients.

The methods also comprise administering an antioxidant or antimicrobiallung surfactant protein compound along with a lipophilic solvent orcarrier. The lipophilic solvent or carrier may be an organic solvent,phosphatidylcholine, cholesterol, or surfactant phospholipid.

The lung surfactant proteins may be used as ingredients of medicationsor foods to prevent lipid oxidation and/or microbial proliferation ofthose reagents, or as ingredients of cosmetics to prevent spoilage ofthe product or to provide antioxidant or antimicrobial effects on skinand hair. The surfactant proteins may have advantages over antioxidantingredients such as butylated hydroxytoluene (BHT) or antimicrobialingredients such as thimerosal for the preparation of all-naturalproducts, since they are derived from animal sources, are edible and arenontoxic.

A number of novel peptides derived from rat lung surfactant proteinsSP-A and SP-D possess lipid oxidation inhibiting and/or antimicrobialproperties that are similar to the full length molecules. Similarly,novel lipid oxidation suppressant and antimicrobial peptides may bederived from human lung surfactant proteins SP-A and SP-D. Whenadministered by aerosol or by intratracheal instillation, thesetruncated proteins can be used to decrease lung injury due to hyperoxia,inflammation or infection. When included as ingredients in foods,cosmetics and medications, the truncated proteins can inhibit lipidoxidation or microbial proliferation that produces spoilage, off colorsand off flavors. Compared to full length proteins, these peptides aremore easily manufactured in quantities that are practical fortherapeutic use, or as ingredients in foods, cosmetics and medications.Without being limited by theory, however, it is believed thatimmunogenicity problems associated with administration of peptides tohumans should be limited, since they will comprise specific portions ofthe native human lung surfactant protein compound proteins.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating several preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the scope of the invention will become apparentto those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

While the specification concludes with claims particularly pointing outand distinctly claiming the present invention, it is believed that thesame will be understood from the following description taken inconjunction with the accompanying drawings in which:

FIG. 1 depicts the inhibition of copper-induced lipid oxidation by SP-Aand SP-D as measured by a thiobarbituric reactive substances (TBARS)assay;

FIG. 2 depicts the continuous analysis of the antioxidant functions ofSP-A and SP-D and the inhibition of LDL oxidation as measured by aspectrophotometer;

FIG. 3 depicts the structural analysis of antioxidant function of SP-Aas quantified by the TBARS assay;

FIG. 4 depicts the immunoblot analysis of protein carbonyl derivativesformed during copper mediated LDL oxidation;

FIG. 5 depicts the protection from tert-butyl hydroperoxide (t-BOOH)induced cell death by SP-A and SP-D;

FIG. 6 depicts the N-terminal domains of SP-A as related to GBSclearance;

FIG. 7 depicts the attenuation of light scattering by growing E. coli inthe presence of SP-A or SP-D;

FIG. 8 depicts the inhibition of E. coli growth in the presence of SP-Aor SP-D;

FIG. 9 depicts the inhibition of E. coli growth in the presence of SP-Aregarding the N-terminal domain;

FIG. 10 depicts the protection of E. coli from growth inhibition in thepresence of SP-A and SP-D by the outer membrane porin A;

FIG. 11 depicts SP-A inhibition of protein synthesis in H. capsulatum;

FIG. 12 depicts the inhibition and reversal of Ca²⁺ dependentaggregation of unilamellar liposomes composed of E. coli lipids in thepresence of SP-A; and

FIG. 13 depicts the inhibition of AIBN-induced lipid peroxidation bySP-A and SP-D regarding complex formation with copper.

DETAILED DESCRIPTION OF THE INVENTION

The hydrophilic surfactant proteins, SP-A and SP-D, according to thepresent invention directly protect surfactant phospholipids and alveolarmacrophages from oxidative modification and injury and directly inhibitmicrobial growth. The antioxidant and antimicrobial activitiesassociated with SP-A maps to the carboxy terminal domain of the protein,which like SP-D, contains a C-type lectin carbohydrate recognitiondomain (CRD). As a result, surfactant proteins SP-A and SP-D, which areubiquitous among air breathing organisms, contribute to the protectionof the lung from oxidative stresses due to atmospheric or supplementaloxygen, air pollutants and lung inflammation, while further protectingthe lung from inhaled organisms.

The hydrophilic surfactant proteins, SP-A and SP-D, have potent, directphospholipid antioxidant properties. In particular, they show thecapability to prevent and/or to delay the oxidative modification ofsurfactant phospholipids and LDL. Additionally, the inventors of thepresent invention believe, without being limited by theory, that themechanism of action of the antioxidant function competes with the chainof propagation of the lipid peroxidation through an effective scavengingof the peroxylic radicals.

Additionally, SP-A and SP-D, have been found to have potent, macrophageindependent antimicrobial properties. In particular, they show thecapability to prevent and/or to delay the proliferation of variousspecies of E. coli, P. aeruginosa, and the dimorphic fungus HistoplasmaCapsulatum.

The hydrophilic surfactant proteins, and derivatives, analogs, homologs,salts and fragments thereof, as well as lipid oxidation inhibiting andantimicrobial peptides, which substantially correspond in sequence tothe amino acid sequence found in specific portions of SP-A or SP-D, canfind useful therapeutical application in the treatment of theinflammatory and hyperoxic lung disease, atherosclerosis or oxidativeinjury to the skin; and in preventing spoilage, off flavors and offcolors in foods and cosmetics. Additionally, the antimicrobialproperties found in the specific portions of SP-A or SP-D, are useful intreating inflammatory and infectious lung diseases, as well preventingspoilage, off flavors and off colors in foods and cosmetics. The presentinvention also provides compositions for lipid oxidation inhibition andmicrobial proliferation in animals, including man

As used herein, “lung surfactant protein compounds” is intended to referto proteins found in lung surfactant, and includes derivatives, analogs,homologs, salts and fragments thereof. Antioxidant lung surfactantprotein compounds are lung surfactant protein compounds and derivatives,analogs, homologs, salts and fragments thereof, which have antioxidantproperties. Preferably, the antioxidant lung surfactant proteincompounds have phospholipid antioxidant properties. Antioxidant lungsurfactant protein compounds include lipid oxidation inhibitingpeptides, also referred to as antioxidant peptides, which substantiallycorrespond in sequence to amino acid sequence found in specific portionsof antioxidant lung surfactant protein compounds.

As used herein, “antimicrobial lung surfactant protein compounds” refersto lung surfactant protein compounds and derivatives, analogs, homologs,salts and fragments thereof, which have antimicrobial properties.Antimicrobial lung surfactant protein compounds include antimicrobialpeptides which are defined as synthetic chains of up to 100 amino acidsand fragments (portions of the native proteins), which substantiallycorrespond in sequence to amino acid sequence found in specific portionsof antimicrobial lung surfactant protein compounds.

Suitable lung surfactant protein compounds of the present invention aresurfactant proteins A and D. Surfactant proteins A and D are members ofthe collectin family of preimmune opsonins that are thought to play arole in opsonization and clearance of pathogenic microorganisms. Acomprehensive disclosure of pulmonary surfactants A and D may be foundin: (Bridges J P, Damodarasamy M, Kuroki Y, Howles G, Hui D Y, McCormackF X. Pulmonary surfactant proteins A and D are potent endogenousinhibitors of lipid peroxidation and cellular injury, J Biol Chem275:38848-38855, 2000), herein incorporated by reference in itsentirety. All members of this family have similar overall structuralorganization including an N-terminal region containing two interchaindisulfide bonds, a collagen-like region of gly-x-y repeats containinghydroxylated amino acids, an amphipathic helical neck region and aC-terminal, C-type carbohydrate recognition domain (CRD). Trimerizationoccurs by triple helix formation in the collagen-like domain and bundledalpha-helical coiled-coil formation in the neck region. SP-A assemblesinto a hexamer of trimers that are disulfide-linked at the N-terminusand laterally associated through the first portion of the collagen-likedomain, forming a flower bouquet-like structure. SP-D forms a cruciformshaped oligomer of four trimers that are joined by disulfide bonds atthe N-terminus. Experimental data suggest that SP-A protects surfactantfrom serum protein inhibitors, and contributes to the assembly andstability of surfactant membranes and the surfactant aggregate, tubularmyelin. Both SP-A and SP-D appear to participate in host defense againstdiverse microbial species including bacteria, viruses, and fungi.

Suitable peptides of the present invention include peptides whichcorrespond to specific areas of the lung surfactant protein compound andcomprise at least a 93 amino acid sequence derived from the aminoterminal portion of the mature lung surfactant protein compounds. Largerpeptides of from about 148 to about 248 amino acids, each containingwithin its sequence the aforementioned repeat sequence, are alsocontemplated by the present invention.

In one embodiment an antioxidant lung surfactant protein compound orantimicrobial lung surfactant protein compound is a peptide comprisingat least about 93, preferably at least about 96 amino acids. In oneembodiment the antioxidant or antimicrobial lung surfactant proteincompound is a peptide comprising from about 93 to about 248 amino acids,preferably from about 93 to about 148 amino acids, while in anotherembodiment the antioxidant or antimicrobial lung surfactant proteincompound is a peptide comprising from about 96 to about 154 amino acids.In yet another embodiment the antioxidant or antimicrobial lungsurfactant protein compound is a peptide comprising from about 5 toabout 90, preferably from about 5 to about 30, amino acids. Furthermore,in still another embodiment, the antimicrobial lung surfactant proteincompound is a peptide comprising from about 96 to about 375 amino acids(for SP-D).

Preferred antioxidant and/or antimicrobial lung surfactant proteincompounds include peptides which substantially correspond in sequence tothe portions of SP-A and SP-D which map to the antioxidant activityand/or the antimicrobial properties. In one embodiment the antioxidantor antimicrobial lung surfactant protein compound is selected frompeptides which substantially correspond in sequence to the C-type lectincarbohydrate recognition domain of SP-A and peptides which substantiallycorrespond in sequence to the C-type lectin carbohydrate recognitiondomain of SP-D.

In another embodiment the antioxidant or antimicrobial lung surfactantprotein compound is selected from peptides which comprise at least a 93amino acid sequence derived from the carboxyl terminal portion of SP-Aor SP-D.

The peptides of the present invention may be linked to an additionalsequence of amino acids by either or both the N-terminus and theC-terminus, wherein the additional sequences are from 1 to about 45amino acids in length. Such additional amino acid sequences, or linkersequences can be conveniently affixed to a detectable label or solidmatrix, or carrier. Labels, solid matrices and carriers that can be usedwith peptides of the present invention are described below. Typicalamino acid residues used for linking are tyrosine, cysteine, lysine,glutamic acid and aspartic acid, or the like.

Lipid oxidation suppressant peptides and antimicrobial peptides derivedfrom rat lung surfactant proteins or from human lung surfactant proteinspossess lipid oxidation inhibiting or antimicrobial properties that aresimilar to the full length molecules. Suitable peptides are listedbelow, and are compared to the native molecule.

Surfactant Protein A

A1. Neck and CRD of rat SP-A (148 aa)

(SEQ ID NO:2) AYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVSTNGQSVNFDTIKEMCTFRAGGNIAVPRTPEENEAIASIAKKYNNYVYLGMIEDQTPGDFHYLDGASVNYTNWYPGEPRGQGKEKCVEMYTDGTWNDRGCLQYRLAVCEF

A2. Smaller active fragment of rat SP-A (93 aa)

(SEQ ID NO:3) AYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVSTNGQSVNFDTIKEMCTFRAGGNIAVPRTPEENEAIASIAKKYNNYVYLGMIEDQ

A3. An active synthetic peptide derived from rat sequence (21 aa) ThrPro Gly Asp Phe His Tyr Leu Asp Gly Ala Ser Val Asn Tyr Thr Asn Trp TyrPro Gly (SEQ ID NO:16)

A4. Human SP-A (248 aa)

(SEQ ID NO:4) MWLCPLALTLILMAASGAACEVKDVCVGSPGIPGTPGSHGLPGRDGRDGVKGDPGPPGPMGPPGETPCPPGNNGLPGAPGVPGERGEKGEPGERGPPGLPAHLDEELQATLHDFRHQILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACARAGGRIAVPRNPEENEAIASFVKKYNTYAYVGLTEGPSPGDFRYSDGTPVNYTNWYRGEPAGRGKEQCVEMYTDGQWNDRNCLYSRLTICEF

A5. Active region of human SP-A-Neck and CRD of human SP-A (148 aa)

(SEQ ID NO:5) AHLDEELQATLHDFRHQILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACARAGGRIAVPRNPEENEAIASFVKKYNTYAYVGLTEGPSPGDFRYSDGTPVNYTNWYRGEPAGRGKEQCVEMYTDGQWNDRNCLYSRLTICEF

A6. An active fragment of human SP-A based on the rat SP-A data (93 aa)

(SEQ ID NO:6) AHLDEELQATLHDFRHQILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACARAGGRIAVPRNPEENEAIASFVKKYNTYAYVGLTEGP

A7. An active synthetic peptide derived from human sequence (21 aa)

(SEQ ID NO:17) Ser Pro Gly Asp Phe Arg Tyr Ser Asp Gly Thr Pro Val AsnTyr Thr Asn Trp Tyr Arg GlySurfactant Protein D

D1. Full length rat SP-D (374 aa)

(SEQ ID NO:7) MLHFLSMLVLLVQPLGDLGAEMKTLSQRSITNTCTLVLCSPTENGLPGRDGRDGREGPRGEKGDPGLPGPMGLSGLPGPRGPVGPKGENGSAGEPGPKGERGLVGPPGSPGISGPAGKEGPSGKQGMGPQGKPGPKGEAGPKGEVGAPGMQGSAGAKGPAGPKGERGAPGEQGAPGNAGAAGPAGPAGPQGAPGSRGPPGLKGDRGAPGDRGIKGESGLPDSAALRQQMEALNGKLQRLEAAFSRYKKAALFPDGQSVGDKLFRAANSEEPFEDAKEMCRQAGGQLASPRSATENAAVQQLVTAHSKAAFLSMTDVGTEGKFTYPTGEALVYSNWAPGEPNNNGGAENCVEIFTNGQWNDKACGEQRLVICEF

D2. Neck and CRD of rat SP-D (the final 153 aa)

(SEQ IDNO:8) DSAALRQQMEALNGKLQRLEAAFSRYKiKAALFPDGQSVGDKIFRAANSEEPFEDAKEMCRQAGGQLASPRSATENAAVQQLVTAHSKAAFLSMTDYGTEGKFTYPTGEALVYSNWAPGEPNNNGGAENCVEJFTNGQWNDKACGEQRLV ICEF

D3. Human SP-D (375 aa)

(SEQ ID NO:9) MLLFLLSALV LLTQPLGYLE AEMKTYSHRT MPSACTLVMC SSVESGLPGRDGRDGREGPRGEKGDPGLPG AAGQAGMPGQ AGPVGPKGDN GSVGEPGPKG DTGPSGPPGPPGVPGPAGREGALGKQGNLG PQGKPGPKGE AGPKGEVGAP GMQGSAGARG LAGPKGERGVPGERGVPGNTGAAGSAGAMG PQGSPGARGP PGLKGDKGIP GDKGAKGESG LPDVASLRQQVEALQGQVQHLQAAFSQYKK VELFPNGQSV GEKIFKTAGF VKPFTEAQLL CTQAGGQLASPRSAAENAAL QQLVVAKNEA AFLSMTDSKT EGKFTYPTGE SLVYSNWAPGEPNDDGGSEDCVEIFTNGKWNDRACGEKRLVVCEF

D4. Neck and CRD of human SP-D (the final 154 aa)

(SEQ ID NO:10) DVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSGEKJFKTAGFVKPFTEAQLLCTQAGGQLASPRSAAENAALQQLVVAKNEAAFLSMTDSKTEGKFTYPTGESLVYSNWAPGEPNDDGGSEDCVEIFTNGKWNDRA CGEKRLVVCEF

D5. An active fragment of human SP-D based on the rat SP-A data (96 aanear the C-terminus of the molecule)

(SEQ ID NO:11) DVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSVGEKJFKTAGFVKPFTEAQLLCTQAGGQLASPRSAAENAAIQQLVVAKNEAAYLSMTDS

In one embodiment of the invention, the lipid oxidation inhibitingpeptides and antimicrobial peptides of the present inventionsubstantially correspond to peptides having the amino acid sequences setforth in SEQ ID NOS:2-11 and 16-17, preferably SEQ ID NOS:4-6, 9-11 and17.

In another embodiment of the invention, the lipid oxidation inhibitingpeptides and the antimicrobial peptides of the present inventionsubstantially correspond to the following amino acid sequences:

(SEQ ID NO:12) MSLCSLAFTLFLTVVAGIKCNVTDVCAGSPGIPGAPGNHGLPGRDGRDGVKGDPGPPGPMGPPGGMPGLPGRDGLPGAPGAPGERGDKGEPGERGLPGFPAYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVFSTNGQSVNFDTIKEMCTRAGGNIAVPRTPEENEAIASJAKKYNNYVYLGMIEDQTPGDFHYLDGASVNYTNWYPGEPRGQGKEKCVEMYTDGTWNDRGCLQYRLAVCEF (SEQ ID NO:13)AYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVFSTNGQSVNFDTIKEMCTRAGGNIAVPRTPEENEAIASIAKKYNNYVYLGMIEDQTPGDFHYLDGASVNYTNWYPGEPRGQGKEKCVEMYTDGTWNDRGCLQYRLAYCEF (SEQ ID NO:14)MSLCSLAFTLFLTVVAGIKCNVTDVCAGSPGIPGAPGNHGLPGRDGRDGVKGDPGPPGPMGPPGGMPGLPGRDGLPGAYGAYGERGDKGEPGERGLPGFPAYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVFSTNGQSVNFDTJKEMCTRAGGNJAVPRTPEENEAIASIAKKYNNYVYLGMIED (SEQ ID NO:15)AYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVFSTNGQSVNFDTIKEMCTRAGGNIAVPRTPEENEAIASIAKKYNNYVYLGMIEDas well as homologs and analogs thereof; wherein:

A = Ala = Alanine R = Arg = Arginine N = Asn = Asparagine D = Asp =Aspartic acid B = Asx = Asparagine or aspartic acid C = Cys = Cysteine Q= Gin = Glutamine E = Glu = Glutamic acid Z = Glx = Glutamine orGlutamic acid G  Gly = Glycine H = His = Histidine I = Ile = IsoleucineL = Leu = Leucine K = Lys = Lysine F = Phe = Phenylalanine P = Pro =Proline S = Ser = Serine T = Thr = Threonine W = Trp = Tryptophan Y =Tyr = Tyrosine V = Val = Valine

The one-letter symbols used to represent the amino acid residues in thepeptides of the present invention are those symbols commonly used in theart.

In another embodiment of the invention, an antioxidant or antimicrobialpeptide of the present invention substantially corresponds to the aminoacid sequence selected from the group consisting of:

(SEQ ID NO:1) Met-Phe-Leu-Lys-Ala-Val-Val-Leu-Thr-Val-Ala-Leu-Val-Ala-IUe-Thr-Gly-Thr-GIn-Ala-GLu-VaI-Thr-Ser-Asp-Gln-Val-Ala-Asn-Val; (SEQ ID NO:16) Thr Pro Gly Asp Phe His Tyr LeuAsp Gly Ala Ser Val Asn Tyr Thr Asn Trp Tyr Pro Gly; (SEQ ID NO:17) SerPro Gly Asp Phe Mg Tyr Ser Asp Gly Thr Pro Val Asn Tyr Thr Asn Trp TyrMg Gly;and mixtures thereof.

By “substantially corresponding” is meant an amino acid sequence havinga homology to any of the listed sequences of about 70%.

As used herein, “peptide” refers to a linear series of amino acidresidues connected to one another by peptide bonds between thealpha-amino and carboxy groups of adjacent amino acid residues. The term“synthetic peptide” is intended to refer to a chemically derived chainof amino acid residues linked together by peptide bonds and which isfree of naturally occurring proteins and fragments thereof.Additionally, analogs, homologs, fragments, chemical derivatives andpharmaceutically acceptable salts of the novel peptides provided hereinare included within the scope of the term “peptide”. In one embodimentof the invention an antioxidant or antimicrobial peptide comprises lessthan about 100 amino acid residues.

By “peptide analog” is meant a peptide which differs in amino acidsequence from the native peptide only by conservative amino acidsubstitutions, for example, substitution of Leu for Val, or Arg for Lys,etc., or by one or more non-conservative amino acid substitutions,deletions, or insertions located at positions which do not destroy thebiological activity of the peptide (in this case, the ability of thepeptide to target vascular lesions). A peptide analog, as used herein,may also include, as part or all of its sequence, one or more amino acidanalogs, molecules that mimic the structure of amino acids, and/ornatural amino acids found in molecules other than peptide or peptideanalogs.

By “homologs” is meant the corresponding peptides derived from otherknown antioxidant or antimicrobial lung surfactant protein compoundproteins having the same or substantially the same properties.

By “analogs” is meant substitutions or alterations in the amino acidsequences of the peptides of the invention, which substitutions oralterations do not abolish the antioxidant or antimicrobial propertiesof the peptides. Thus, an analog might comprise a peptide having asubstantially identical amino acid sequence to a peptide provided hereinas SEQ ID NO: 1 wherein one or more amino acid residues have beenconservatively substituted with chemically similar amino acids. Examplesof conservative substitutions include the substitution of a non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another. Likewise, the present invention contemplates thesubstitution of one polar (hydrophilic) residue such as between arginineand lysine, between glutamine and asparagine, and between glycine andserine. Additionally, the substitution of a basic residue such aslysine, arginine or histidine for another or the substitution of oneacidic residue such as aspartic acid or glutamic acid for another isalso contemplated.

Analogs may also encompass additional amino acids, added to the N-and/or C-terminal portion of the peptide. For example, analogs of thepeptides of the invention may contain cysteine or another amino acid, atthe amino or carboxyl end of the peptide by which the peptide may becovalently attached to a carrier protein, e.g., albumin, for in vivoadministration. Other carrier molecules include polyethylene glycol(PEG) which functions to avoid proteolytic cleavage and clearing ofpeptides from the blood.

The phrase “conservative substitution” also includes the used ofchemically derivatized residues in place of a non-derivatized residuesas long as the peptide retains the requisite antioxidant orantimicrobial properties. Analogs also include the presence ofadditional amino acids or the deletion of one or more amino acids thatdo not affect biological activity. For example, analogs of the subjectpeptides may contain an N- or C-terminal cysteine, by which, if desired,the peptide may be covalently attached to a carrier protein, e.g.,albumin. Such attachment, it is believed, will minimize clearing of thepeptide from the blood and also prevent proteolysis of the peptides.)

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,protein chemistry, molecular biology, microbiology, and recombinant DNAtechnology, which are well within the skill of the art. Such techniquesare explained fully in the literature.

In preferred embodiments, the peptide or peptide analog is watersoluble; or is soluble in a physiological fluid, preferably, one that isat physiological pH, for example, blood plasma.

In another preferred embodiment, the peptide has a molecularconformation analogous to the molecular conformation (size, shape,charge) of a surface region of the lung surfactant protein compoundsmoiety. Examples of peptides believed to have a molecular conformationanalogous to the molecular conformation of a surface region of the lungsurfactant protein include:

(SEQ ID NO:1) Met-Phe-Leu-Lys-Ala-Val-Val-Leu-Thr-Val-Ala-Leu-Val-Ala-Ile-Thr-Gly-Thr-Gln-Ala-Glu-Val-Thr-Ser-Asp-Gln-Val-Ala-Asn-Val; (SEQ ID NO:16) Thr Pro Gly Asp Phe His Tyr LeuAsp Gly Ala Ser Val Asn Tyr Thr Asn Trp Tyr Pro Gly; (SEQ ID NO:17) SerPro Gly Asp Phe Arg Tyr Ser Asp Gly Thr Pro Val Asn Tyr Thr Asn Trp TyrArg Gly;and derivatives, analogs, homologs, fragments, salts and mixturesthereof.

In various other preferred embodiments, the peptide or peptide analoghas an acetylated amino terminus and/or an amidated carboxy terminus.Examples of such peptide or peptide analogs include:

(SEQ ID NO:1) H₂N--Met-Phe-Leu-Lys-Ala-Val-Val-Leu-Thr-Val-Ala-Leu-Val-Ala-Ile-Tbr-Gly-Thr-Gln-Ala-Glu-Val-Thr-Ser-Asp-Gln-Val-Ala-Asn-Val--CONH₂; (SEQ ID NO:16) H₂N--Thr Pro Gly AspPhe His Tyr Leu Asp Gly Ala Ser Val Asn Tyr Thr Asn Trp Tyr ProGly--CONH₂; (SEQ ID NO:17) H₂N--Ser Pro Gly Asp Phe Arg Tyr Ser Asp GlyThr Pro Val Asn Tyr Thr Asn Trp Tyr Arg Gly--CONH₂;and derivatives, analogs, homologs, fragments, salts and mixturesthereof.

By “derived from” is meant having an amino acid sequence identical orsubstantially identical to the sequence of, as used herein, asurfactant-associated protein. By “substantially identical to” is meanthaving an amino acid sequence that differs only by conservative aminoacid substitutions or by non-conservative amino acid substitutions,deletions, or insertions located at positions that do not destroy thebiological activity of the peptide.

It is possible to design any number of peptide analogs, having differentamino acid sequences, provided that the local charge distribution (andoverall net charge) and secondary structure, and hence the biologicalactivity is maintained. Such peptide analogs will generally differ fromthe native protein sequences by conservative amino acid substitutions(e.g., substitution of Leu for Val, or Arg for Lys, etc.) well known tothose skilled in the art of biochemistry.

The peptides, once designed, can be synthesized by any of a number ofestablished procedures, including, e.g., the expression of a recombinantDNA encoding that peptide in an appropriate host cell. Alternatively,these peptides can be produced by the established procedure of solidphase peptide synthesis. Briefly, this procedure entails the sequentialassembly of the appropriate amino acids into a peptide of a desiredsequence while the end of the growing peptide is linked to an insolublesupport. Usually, the carboxyl terminus of the peptide is linked to apolymer from which it can be liberated upon treatment with a cleavagereagent. The peptides so synthesized are then labeled with a reagentthat enables the monitoring of the peptide after its administration to apatient. Finally, the SP-A and SP-D may be purified from natural sourcesincluding, but not limited to, human.

Preferably the synthesized peptide substantially corresponds to theamino acid sequence of an antioxidant or antimicrobial lung surfactantpeptide, or to a portion of an antioxidant or antimicrobial lungsurfactant peptide which maps to antioxidant or antimicrobial activity.As used herein, the term “substantially corresponds” means a peptideamino acid sequence having approximately 70% homology in amino acidsequence to an antioxidant or antimicrobial lung surfactant peptide.

The term “chemical derivative” is meant to include any peptide derivedfrom a peptide of the present invention wherein one or more amino acidshave been chemically derivatized by reaction of one or more functionalside groups of the amino acid residues present in the peptide. Thus, a“chemical derivative” as used herein is a peptide that is derived fromthe peptides identified herein by one or more chemical steps. Examplesof derivatized molecules include molecules where free amino groups havebeen derivatized to form amine hydrochlorides, p-toluene sulfonylgroups, carbobenzoxy groups, t-butyloxycarbonyl groups,thiourethane-type derivatives, trifluroroacetyl groups, chioroacetylgroups or formyl groups. Free carboxyl groups may be derivatized to formsalts, methyl and ethyl esters or other types of esters or hydrazides.

Free hydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-imbenzylhistidine. Also included as chemical derivatives arethose peptides that contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine.

The term “fragment” refers to any subject peptide having an amino acidsequence shorter than that of a peptide provided herein as any of SEQ IDNOS: 1-17 wherein the fragment retains antioxidant or antimicrobialproperties of the subject peptides. The peptides of the presentinvention, homologs and analogs thereof may be synthesized by a numberof known techniques. For example, the peptides may be prepared using thesolid-phase synthetic technique or other peptide synthesis techniqueswell known to those skilled in the art. The peptides of the presentinvention might also be prepared by chemical or enzymatic cleavage fromlarger portions of the lung surfactant protein compound or from theentire lung surfactant molecule.

Additionally, the peptides of the present invention may also be preparedby recombinant DNA techniques. For most of the amino acids used to buildproteins, more than one coding nucleotide triplet (codon) can code for aparticular amino acid residue. This property of the genetic code isknown as redundancy. Therefore, a number of different nucleotidesequences may code for a particular subject eating suppressant peptide.The present invention also contemplates a deoxyribonucleic acid (DNA)molecule or segment that defines a gene coding for, i.e., capable ofexpressing, a subject polypeptide or a subject chimeric polypeptide fromwhich a polypeptide of the present invention may be enzymatically orchemically cleaved.

DNA molecules that encode the subject peptides can be synthesized bychemical techniques, for example, the phosphotriester method ofMatteuccie et al., Chem. Soc. 103:3185 (1981). Using a chemical DNAsynthesis technique, desired modifications in the peptide sequence canbe made by making substitutions for bases that code for the native aminoacid sequence. Ribonucleic acid equivalents of the above-described DNAmolecules may also be used.

A nucleic acid molecule comprising a vector capable of replication andexpression of a DNA molecule defining coding sequence for a subjectpolypeptide or subject chimeric polypeptide is also contemplated.

The peptides of the present invention are preferably chemicallysynthesized by the Merrifield solid phase technique. In general, themethod comprises the sequential addition of one or more amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group such as lysine.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid andthe like; and organic acids such as formic acid, acetic acid, propionicacid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonicacid, succinic acid, maleic acid, fumaric acid, anthranilic acid,cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.

Suitable bases capable of forming salts with the subject peptidesinclude inorganic bases such as sodium hydroxide, ammonium hydroxide,potassium hydroxide and the like; and organic bases such as mono-,di-and tri-alkyl amines (e.g., triethyl amine, diisopropyl amine, methylamine, dimethyl amine and the like) and optionally substitutedethanolamines (e.g. ethanolamine, diethanolamine and the like).

The peptides of the present invention may be synthesized using anautomatic solid phase peptide synthesizer.

Antioxidants or antimicrobial lung surfactant protein compounds, whichinclude lung surfactant protein compounds and derivatives, analogs,homologs, salts and fragments thereof which have antioxidant orantimicrobial properties, may be used therapeutically. Preferredtherapeutic antioxidant or antimicrobial lung surfactant proteincompounds are lipid oxidation inhibiting or antimicrobial peptides.

It is understood that the present invention is not limited by anyparticular theory or proposed mechanism to explain its effectiveness inan end-use application.

Therapies

Lung injury, such as pneumonia, is a disease state characterized by thedevelopment or persistence of pulmonary infiltrates, reduced lungcompliance and reduced gas exchange. The identification of thosepatients who are in need of treatment for acute lung injury is wellwithin the ability and knowledge of one of ordinary skill in the art.For example, individuals who are either suffering from clinicallysignificant acute lung injury (ALI) or who are at risk of developingclinically significant acute lung injury are patients in need oftreatment. A clinician of ordinary skill in the art can readilydetermine, by the use of clinical tests, physical examination andmedical/family history, if an individual is a patient in need oftreatment for acute lung injury.

Antioxidant and antimicrobial lung surfactant protein compounds inaccordance with the present invention may be used in treating acute lunginjury, including adult respiratory distress syndrome and hyperoxic lunginjury. In one embodiment a patient with adult respiratory distresssyndrome or hyperoxic lung injury is treated with SP-A and/or SP-D, or apeptide which corresponds to the portions of SP-A and/or SP-D which mapto antioxidant or antimicrobial activity. In another embodiment, thepatient is treated with a peptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 1-17, preferably SEQ ID NOS: 1,4-6, 9-15 and 17. Generally the patient is treated with an amount ofantioxidant or antimicrobial lung surfactant protein compound that iseffective, upon single or multiple dose administration to the patient,in providing relief of symptoms associated with acute lung injury.

An effective dose is an amount that is effective in inhibiting thedevelopment or persistence of acute lung injury in a patient in needthereof. As such, successful treatment of a patient for acute lunginjury is understood to include effectively slowing, interrupting,arresting, or stopping acute lung injury and/or pneumonia and does notnecessarily indicate a total elimination of acute lung injury. It isappreciated by those of ordinary skill in the art that successfultreatment for ALI can include prophylaxis in preventing ALI.

Peroxidation of surfactant lipids, such as the unsaturated fatty acidportions of surfactant cholesteryl lipids and surfactant phospholipids,is known to cause surfactant dysfunction. The identification of thosepatients who are in need of inhibition of peroxidation of surfactantlipids is well within the ability and knowledge of one of ordinary skillin the art. For example, those individuals who are in need of treatmentfor acute lunge injury, as defined above, are also patients who are inneed of inhibition of peroxidation of surfactant lipids. A preferredantioxidant or antioxidation amount is an amount sufficient forinhibiting the peroxidation of surfactant lipids in a patient's lung.

As used herein, the term “patient” refers to a warm-blooded animal ormammal which is in need of treatment for a chronic heart disease,atherosclerosis, hypercholesterolemia or which is in need of inhibitingoxidation.

A “therapeutically effective amount” is an amount that is effective,upon single or multiple dose administration to the patient, in providingrelief of symptoms associated with acute lung injury and/or pneumonia.

As used herein, “relief of symptoms” refers to decrease in severity overthat expected in the absence of treatment and does not necessarilyindicate a total elimination or cure of the disease. Relief of symptomsis also intended to include prophylaxis.

In determining the therapeutically effective amount or dose, theeffective antioxidant or antimicrobial amount or dose of an antioxidantor antimicrobial lung surfactant protein compound, a number of factorsare considered by the attending diagnostician, including, but notlimited to: the species of the mammal; its size, age, and generalhealth; the response of the individual patient; the particular compoundadministered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A therapeutically effective amount will generally vary from about 1milligram per kilogram of body weight per day (mg/kg/day) to about 5grams per kilogram of body weight per day (gm/kg/day). A daily dose offrom about 1 mg/kg to about 500 mg/kg is preferred.

Antioxidant and antimicrobial peptides in accordance with the inventionmay also be used in the treatment of atherosclerosis and systemicinfections. Atherosclerosis is a disease state characterized by thedevelopment and growth of atherosclerotic lesions or plaque.Peroxidation of LDL lipid, such as the unsaturated fatty acid portionsof LDL cholesteryl esters and phospholipids, is known to facilitate thedeposition of cholesterol in macrophages which subsequently aredeposited in the vessel wall and are transformed into foam cells. It isappreciated by those of ordinary skill in the art that successfultreatment for atherosclerosis can include prophylaxis in preventingatherosclerotic lesion or plaque formation. Systemic infections comprisedisease states characterized by the infection in the blood stream ormajor body cavities or organs. It is appreciated by those of ordinaryskill in the art that successful treatment for systemic infections caninclude prophylaxis in preventing sepsis. Local infections, such asskin, wound, eye, mucosal, ear, or perineal infections may also betreated with topical preparations containing surfactant proteins orpeptides.

In effecting treatment of a patient, an antioxidant or antimicrobiallung surfactant protein compound, or any derivative, analog, homolog,salt, fragment or mixture thereof, can be administered in any form ormode which makes the compound bioavailable in effective amounts.Suitable modes of administration include oral, topical, rectal enteralor parenteral administration. Parenteral administration may includeintratracheal or inhalant aerosol administration, transdermaladministration, subcutaneous injection, intravenous injection,intraperitoneal injection, intramuscular injection, intrasternalinjection, intrathecal injection, intraventricular andintracerebroventricular injection and infusion techniques. Intratrachealadministration or inhalation of aerosolized compositions are generallypreferred. One skilled in the art of preparing formulations can readilyselect the proper form and mode of administration depending upon therelevant circumstances.

An antioxidant or antimicrobial lung surfactant protein compound,derivative, analog, homolog, fragment, salt or mixtures thereof can beadministered in the form of pharmaceutical compositions or medicamentswhich are made by combining an antioxidant or antimicrobial lungsurfactant protein compound, or any derivative, analog, homolog, salt,fragment or mixture thereof, with pharmaceutically acceptable carriersor excipients, the proportion and nature of which are determined by thechosen route of administration, and standard pharmaceutical practice.The term “pharmaceutically acceptable” refers to a molecular entity orcomposition that does not produce an allergic or similar unwantedreaction when administered to animals or humans. Preferred carriersinclude natural and synthetic surfactant phospholipids.

The pharmaceutically acceptable carriers used in conjunction with thepeptides of the present invention vary according to the mode ofadministration. For example, the compositions may be formulated in anysuitable carrier for oral liquid formulation such as suspensions,elixirs and solutions. Compositions for liquid oral dosage include anyof the usual pharmaceutical media such as, for example, water, oils,alcohols, flavoring agents, preservatives, coloring agents and the like.In the case of oral solid preparations (powder capsules and tablets)carriers such as starches, sugars, diluents, granulating agents,lubricants, binders, disintegrating agents and the like may be used. Inaddition, carriers such as liposomes, microemulsions andself-emulsifiable glasses may be used.

The compositions of the present invention may also be formulated forintravenous administration. In this instance, the peptides are admixedwith sterile water and saline or other pharmaceutically acceptablecarrier.

The pharmaceutical compositions or medicaments are prepared in a mannerwell known in the pharmaceutical art. The carrier or excipient may be asolid, semi-solid, or liquid material that can serve as a vehicle ormedium for the active ingredient. Suitable carriers or excipients arewell known in the art. The pharmaceutical composition may be adapted fororal or parenteral use and may be administered to the patient in theform of tablets, capsules, suppositories, solution, suspensions, or thelike.

The pharmaceutical compositions may be administered orally, for example,with an inert diluent or with an edible carrier. They may be enclosed ingelatin capsules or compressed into tablets. For the purpose of oraltherapeutic administration, an antioxidant or antimicrobial lungsurfactant protein compound, derivative, salt or fragment thereof may beincorporated with excipients and used in the form of tablets, troches,capsules, elixirs, suspensions, syrups, wafers, chewing gums and thelike. These preparations should contain at least about 4% of anantioxidant or antimicrobial lung surfactant protein compound,derivative, salt or fragment thereof the active ingredient, but may bevaried depending upon the particular form and may conveniently bebetween 4% to about 70% of the weight of the unit. The amount of theactive ingredient present in compositions is such that a unit dosageform suitable for administration will be obtained.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following adjuvants: binders, such as microcrystallinecellulose, gum tragacanth or gelatin; excipients, such as starch orlactose, disintegrating agents such as alginic acid, Primogel, cornstarch and the like; lubricants, such as magnesium stearate or Sterotex;glidants, such as colloidal silicon dioxide; and sweetening agents, suchas sucrose or saccharin may be added or flavoring agents, such aspeppermint, methyl salicylate or orange flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or a fatty oil. Otherdosage unit forms may contain other various materials that modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills may be coated with sugar, shellac, or other entericcoating agents. A syrup may contain, in addition to the activeingredient, sucrose as a sweetening agent and certain preservatives,dyes and colorings and flavors. Materials used in preparing thesevarious compositions should be pharmaceutically pure and non-toxic inthe amounts used.

For the purpose of parenteral administration, an antioxidant orantimicrobial lung surfactant protein compound, derivative, analog,homolog, fragment, salt or mixtures thereof may be incorporated into asolution or suspension. These preparations should contain at least about0.1% of a compound of the invention, but may be varied to be betweenabout 0.1 and about 50% of the weight thereof. The amount of the activeingredient present in such compositions is such that a suitable dosagewill be obtained.

When administered intravenously, the peptide compositions may becombined with other ingredients, such as carriers and/or adjuvants. Thepeptide can also be covalently attached to a protein carrier, such asalbumin, so as to minimize clearing of the peptides. There are nolimitations on the nature of the other ingredients, except that theymust be pharmaceutically acceptable, efficacious for their intendedadministration and cannot degrade the activity of the active ingredientsof the compositions. The peptide compositions of the invention may alsobe impregnated into transdermal patches or contained in subcutaneousinserts, preferably in a liquid or semi-liquid form that patch or inserttime releases therapeutically effective amounts of one or more of thesubject peptides.

The pharmaceutical forms suitable for administration intravenously orinto the airways include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases the ultimate solution form mustbe sterile and fluid. Typical carriers include a solvent or dispersionmedium containing, for example, water buffered aqueous solutions (i e.,biocompatible buffers), ethanol, polyols such as glycerol, propyleneglycol, polyethylene glycol, suitable mixtures thereof, surfactants orvegetable oils. Sterilization can be accomplished by any art-recognizedtechnique, including but not limited to, filtration or addition ofantibacterial or antifungal agents, for example, paraben, chlorobutanol,phenol, sorbic acid or thimerosal. Further, isotonic agents such assugars or sodium chloride may be incorporated in the subjectcompositions.

Production of sterile injectable solutions containing the subjectpeptides is accomplished by incorporating these compounds in therequired amount in the appropriate solvent with various ingredientsenumerated above, as required, followed by sterilization, preferablyfilter sterilization. To obtain a sterile powder, the above solutionsare vacuum-dried or freeze-dried as necessary.

The solutions or suspensions may also include one or more of thefollowing adjuvants depending on the solubility and other properties ofan antioxidant or antimicrobial lung surfactant protein compound,derivative, salt or fragment thereof: sterile diluents such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants or antimicrobials suchas ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of toxicity such as sodiumchloride or dextrose.

The parenteral preparation can be enclosed in ampules, disposablesyringes or multiple dose vials made of glass or plastic.

The pharmacological composition will preferably comprise one or moreantioxidant or antimicrobial lung surfactant protein compounds thereofalong with a pharmaceutically acceptable carrier, fillers or excipients.The administering step may comprise administering a pharmacologicalcomposition comprising an antioxidant or antimicrobial lung surfactantprotein compound, derivative, salt or fragment thereof along withpharmaceutically acceptable carrier, fillers or excipients.

The methods may be by oral administration of the composition or apharmaceutically acceptable salt or derivative thereof into said mammal.The methods according to the present invention preferable allows theadministration of the antioxidant or antimicrobial molecule isadministered in a unitary dose of from about 1 to about 1000 mg. Aunitary dose is generally administered from about 1 to about 3 times aday.

The administering step may comprise parenteral administration of theantioxidant or antimicrobial compound or a pharmaceutically acceptablesalt or derivative thereof into said mammal. This administration may beby transdermal administration, subcutaneous injection, intravenousinjection, intraperitoneal injection, intramuscular injection,intrasternal injection, intrathecal injection, intracerebroventricularinjection and infusion techniques. Preferred techniques includeintratracheal administration and administration by inhalation ofaerosolized compositions, such as with a nebulized or metered doseinhaler.

Methods in accordance with the present invention also include methodswhich comprise administering antioxidant or antimicrobial compounds or apharmaceutically acceptable salt or derivative thereof along with alipophilic compound, such as a lipophilic solvent or carrier. Thelipophilic solvent or carrier may be an organic solvent,phosphatidylcholine, cholesterol, or surfactant phospholipids.

The pharmaceutical compositions of the present invention can beformulated for the inhaled, intratracheal, oral, sublingual,subcutaneous, intravenous, transdermic or rectal administrations indosage units and in admixture with pharmaceutical excipients orvehicles. Convenient dosage forms include, among those for oraladministration, tablets, powders, granulates, and, among those forparenteral administration, solutions especially for transdermaladministration, subcutaneous injection, intravenous injection,intraperitoneal injection, intramuscular injection, intrasternalinjection, intrathecal injection and infusion techniques.

The dosage can vary widely as a function of the age, weight and state ofhealth of the patient, the nature and the severity of the ailment, aswell as of the administration route. These doses can naturally beadjusted for each patient according to the results observed and theblood analyses previously carried out.

Food Preservatives

The antioxidant or antimicrobial lung surfactant protein compounds ofthe present invention may be made into edible nonionic water-orlipid-soluble additives that are effective antioxidants and/orantimicrobials in food products or ingredients of foods withoutimparting undesirable flavors, aromas, and precipitates.

Oxidation of fats, vegetable oils, carotenoids and their biologicallyactive derivatives, Vitamin A, and of essential oils and otherflavorings results in degradation of their quality, and is deleteriousto foodstuffs containing these products. Bacterial contamination,colonization and/or proliferation in food products and ingredientseasily occurs in various environments.

The art shows many methods of inhibiting lipid oxidation or microbialproliferation by adding fat-soluble antioxidants or by addingantimicrobials to a substrate. The art does not show the oxidation ormicrobial stabilization of fats, oils, foods and ingredients of foodsemploying lung surfactant protein compounds and active derivatives andfragments in a form effective for such purpose.

Methods of preventing oxidation in a lipid-containing food and/orpreventing microbial contamination, colonization, persistence orproliferation in a food comprise incorporating in the food anoxidation-inhibiting amount or antimicrobial amount of one or more lungsurfactant protein compounds, derivatives, analogs, homologs, fragments,salts or mixtures thereof to protect the food from oxidation ormicrobial contamination. An oxidation-inhibiting or antimicrobial amountis an amount sufficient to inhibit lipid oxidation or inhibit microbialgrowth.

The food composition may further comprise carriers, fillers, andexcipients. Preferably, the lung surfactant protein compound makes upabout 0.01% to about 10% of the final weight of the food product. Morepreferably, the lung surfactant protein compound makes up from about0.02% to about 5% of the final weight of the food product.

Further, a fat, oil, fatty food or food ingredient substrate stabilizedagainst oxidation with such composition, such a stabilized substratewherein the substrate includes a carotenoid, and a method of stabilizinga fat, oil, food, or food ingredient substrate which includes the stepof introducing into the substrate such a composition as set forth in theforegoing, and such a method wherein the substrate includes acarotenoid.

Pharmaceutical Compositions

The compositions of the present invention may also be used as a methodof preventing oxidation and/or bacterial proliferation inlipid-containing pharmaceuticals. This embodiment involves incorporatingin the pharmaceutical an oxidation-inhibiting or antimicrobial amount ofone or more lung surfactant protein compounds, or derivative, analog,homolog, fragment, salt or mixture thereof.

The composition may further include carriers, fillers, and excipients.Preferably, the lung surfactant protein compounds makes up about 0.01%to about 25% of the final weight of the pharmaceutical product. Morepreferably, the lung surfactant protein compounds makes up from about0.05% to about 10% of the final weight of the pharmaceutical product.

Cosmetic or Dermatological Preparations

In one embodiment, the present invention provides methods of preventingoxidation in a lipid-containing cosmetic or dermatological compositionby incorporating, in a suitable vehicle containing cosmetic ordermatological composition, an oxidation-inhibiting amount of one ormore antioxidant lung surfactant protein compound, derivative, analog,homolog, fragment, salt or mixtures thereof.

In another embodiment, the present invention provides methods ofpreventing microbial proliferation in a cosmetic or dermatologicalcomposition by incorporating, in a suitable vehicle containing cosmeticor dermatological composition, an antimicrobial amount of one or moreantimicrobial lung surfactant protein compounds, or derivatives,analogs, homologs, fragments, salts or mixtures thereof.

The composition may further comprise carriers, fillers, and excipients.Preferably, the antioxidant or antimicrobial lung surfactant proteincompound is present at a concentration between about 0.005% and about25% by weight with respect to the total weight of the composition. Morepreferably, the lung surfactant protein compound is present at aconcentration between about 0.05% and about 10% by weight with respectto the total weight of the composition.

In another embodiment, the present invention relates to a newantioxidant or antimicrobial system based on one or more lung surfactantprotein compounds, derivative, analog, homolog, fragment, salt ormixtures thereof for use as an antioxidant or antimicrobial system incompositions based on an oleaginous material containing such a systemand, principally, cosmetic compositions.

This embodiment generally provides for a cosmetic or dermatologicalcomposition containing, in a suitable vehicle, an oxidation-inhibitingamount or antimicrobial amount of a lung surfactant protein compound,derivative, analog, homolog, fragment, salt or mixtures thereof.

The cosmetic or dermatological composition may be in the form of asuspension or dispersion in a solvent or a fatty substance, or in theform of an emulsion, or in the form of an ointment, a gel, a solidstick, or an aerosol foam.

The cosmetic or dermatological composition may additionally contain oneor more cosmetic adjuvants such as lower alcohols, polyols, esters of,fatty acids, oils, and waxes, solvents, silicones, thickeners,emollients, UV-A, UV-B and broad band sunscreens, antifoam agents,hydrating agents, perfumes, stabilizers, surfactants, fillers,sequestrants, anionic, cationic, nonionic and amphoteric polymers andmixtures thereof, propellants, alkalifying and acidifying agents, dyesand metal oxide pigments.

Preferably, the lung surfactant protein compound is present at aconcentration between about 0.001% and about 25% by weight with respectto the total weight of the composition. More preferably, the lungsurfactant protein compound is present at a concentration between about0.005% and about 15% by weight with respect to the total weight of thecomposition.

In another embodiment, the present invention relates to an antioxidantor antimicrobial cosmetic system based on lung surfactant proteincompounds or at least one of its derivatives, salts or fragments thereofwhich contains either at least one basic agent or includes at least onetocopherol or a derivative thereof. Preferably the system contains fromabout 0.5 to about 20 weight percent of a tocopherol or derivativethereof, about 0.5 to about 50 weight percent of a basic agent and about0.5 to about 90 weight percent of lung surfactant protein compounds andderivatives, salts and fragments thereof. This system is employed incosmetic or pharmaceutical compositions.

The present invention thus relates to a new antioxidant or antimicrobialsystem based on at least one basic agent characterized by the fact thatthe system also includes at least one tocopherol or tocopherolderivative and lung surfactant protein compound, derivative, analog,homolog, fragment, salt or mixtures thereof.

Sodium hydroxide, triethanolamine, basic amino acid may, for example, beused as the basic agent. By basic amino acid is meant a natural basicamino acid such as, for example, lysine, arginine and histidine, theirisomeric or racemic forms, as well as synthetic basic amino acids andderivatives of natural amino acids. Preferably, in accordance with thepresent invention, lysine or arginine is employed.

By the expression “tocopherol” there is meant not only alpha-tocopherolbut also beta, gamma or delta tocopherol as well as their mixtures.Among the tocopherol derivatives mention can be made of the esters oftocopherol such as tocopherol acetate and tocopherol nicotinate.

According to the invention, the antioxidant or antimicrobial systemcomprises, by weight:

-   -   from about 0.5 to about 40 percent of a tocopherol or a        tocopherol derivative,    -   from about 0.5 to about 50 percent of a basic amino acid, and    -   from about 0.5 to about 90 percent of lung surfactant protein        compounds or derivatives, salts or fragments thereof.

The preferred ratio between the concentration of the basic amino acidand the concentration of the tocopherol ranges from about 1:1 to about1:20.

The compositions according to the invention are provided in the form ofoily solutions, water-in-oil or oil-in-water emulsions, optionallyanhydrous products, lotions or even microdispersions or ionic ornonionic lipid vesicles. They constitute principally milks for the careof the skin, creams (face creams, hand creams, body creams, sunscreencreams, make-up remover creams, foundation creams), foundation fluids,make-up remover milks, sunscreen milks, bath oils, lipsticks, eyelidmake-up, deodorant sticks, etc.

For topical application, the pharmaceutical compositions according tothe invention comprise vehicles and ingredients necessary to provide,for example, the composition in the form of ointments, creams, milks,pomades and oily solutions.

According to a preferred embodiment, the cosmetic or dermopharmaceuticalcompositions are provided in forms intended to be topically applied and,in particular, creams intended for the protection of the lipids of theskin against oxidation.

In the compositions according to the invention, the antioxidant orantimicrobial system is generally present in the composition at a levelsufficient to provide, by weight of the total composition:

-   -   tocopherol or derivative thereof from about 0.05 to about 2%;    -   basic agent from about 0.05 to about 5%; and    -   lung surfactant protein compounds from about 0.05 to about 8%.

The compositions of the invention can also contain active compounds oringredients conventionally employed in compositions mentioned above,such as surface active agents, dyes, perfumes, astringent products,ultraviolet absorbing products, organic solvents, water, etc. Thesecompositions are prepared in accordance with conventional methods.

The compositions of the invention may also contain, in the aqueousphase, various complementary additives such as preserving agents,sequestering agents, gelling agents and the like. The compositions ofthe invention may also contain, in the lipid phase, variouscomplementary additives such as oils, waxes or gums having, for example,emollient or lubricating properties. The compositions are most oftenprovided in milk, cream or gel form, other modes of presentation notbeing excluded.

In another embodiment, the present invention provides a process for thepreparation of the compositions described above, comprising:

(i) mixing (a) a fatty phase, comprising the lipophilic surfactant, thehydrophilic surfactant, and the fatty acid and (b) an aqueous phasecomprising the basic agent and the cosmetically or dermatologicallyactive lung surfactant protein compound by stirring to obtain a mixture;and

(ii) homogenizing the mixture by subjecting the mixture to cavitation.

For cosmetic applications, the compositions of the invention may,moreover, be advantageously used in combination with other compoundsdisplaying retinoid-type activity, with the D vitamins or derivativesthereof, with corticosteroids, with anti-free radical agents, withalpha-hydroxy or alpha-keto acids or derivatives thereof, oralternatively with ion channel blockers, all of these different activeagents.

The present invention therefore also features cosmetic compositionscomprising a carrier that is cosmetically acceptable and suitable for atopical application and lung surfactant protein compounds. Such cosmeticcompositions are advantageously presented in the form of a cream, amilk, a lotion, a gel, microspheres or nanospheres or lipid or polymericvesicles, a soap or a shampoo.

The concentration of the lung surfactant protein compound, derivative,analog, homolog, salt, fragment or mixture thereof in the cosmeticcompositions according to the invention advantageously ranges from about0.001% to about 30%, by weight of total composition.

The medicinal and cosmetic compositions according to the invention may,in addition, contain inert or even pharmacodynamically or cosmeticallyactive additives or combinations of these additives, and, especially:wetting agents; depigmenting agents such as hydroquinone, azelaic acid,caffeic acid or kojic acid; emollients; moisturizing agents such asglycerol, PEG 400, thiamorpholinone and its derivatives or alternativelyurea; antiseborrhoeic or antiacne agents such asS-carboxymethylcysteine, S-benzylcysteamine, salts or derivativesthereof, benzoyl peroxide; antibiotics such as erythromycin and estersthereof, neomycin, clindamycin and esters thereof, tetracyclines;antifungal agents such as ketoconazole or4,5-polymethylene-3-isothiazolidones; agents promoting hair regrowth,such as Minoxidil (2,4-diamino-6-piperidinopyrimidine 3-oxide) andderivatives thereof, Diazoxide(7-chloro-3-methyl-1,2,4-benzothiadiazime-1,1-dioxide) and Phenytoin(5,4-diphenyl-2,4-imidazolidinedione); non-steroidal anti-inflammatoryagents; carotenoids and especially beta-carotene; anti-psoriatic agentssuch as anthralin and derivatives thereof; and, lastly,5,8,11,14-eicosatetraynoic and 5,8,11-eicosatrynoic acids and esters andamides thereof.

The compositions according to the invention may also contain taste- orflavor-enhancing agents, preservatives such as parahydroxybenzoic acidesters, stabilizing agents, moisture regulating agents, pH regulatingagents, osmotic pressure modifying agents, emulsifying agents, UV-A andUV-B screening agents, antioxidants and antimicrobials such asalpha-tocopherol, butylated hydroxyanisole or butylated hydroxytoluene.

The following examples illustrate and explain the present invention butshould not be taken as limiting the present invention in any regard.

EXAMPLES

Methods

Purification and modification of surfactant proteins-antioxidantproperties

Native SP-A and SP-D were isolated from the alveolar wash of rats whichhad been pretreated with intratracheal silica to enhance the collectinyield, in accordance with the method of Dethloff et al., Biochem. J.233:111-118 (1986). After centrifugation, rat SP-D was purified bymaltose-Sepharose affinity chromatography of the supernatant and ratSP-A was purified from the pellet by NaBr flotation, butanol extractionand mannose-Sepharose affinity chromatography, in accordance with themethod of McCormack et al., J. Biol. Chem. 272:27971-27979 (1997). MouseSP-D was used interchangeably with rat SP-D. All proteins wereextensively dialyzed to remove residual EDTA. For some experiments, ratSP-A and rat SP-D were alkylated by incubation with 0.5 M iodoacetamideat 37° C. in the dark for 1 hr and then extensively dialyzed. The wildtype and mutant recombinant SP-A, SP-D and MBP used in this study weresynthesized using baculovirus vectors and purified bycarbohydrate-Sepharose affinity chromatography, as previously describedin McCormack et al., J. Biol. Chem. 272:27971-27979 (1997); McCormack etal., Biochemistry 36:13963-13971 (1997); and McCormack et al., J. Biol.Chem. 274:3173-3183 (1999).

Lipid preparations

Substrates for lipid oxidation included mixtures of natural andsynthetic glycerophospholipids that are found in pulmonary surfactantand human LDL. The surfactant lipid mix, composed of eggphosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol and1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (1:1:0.15:0.15, w/w,respectively) (Avanti Polar Lipids) in chloroform, was dried to a filmunder nitrogen. Following hydration in PBS or 0.15 M NaCl for 30 min,multilamellar vesicles were generated by vigorous vortexing for 5minutes. LDL were isolated from the plasma of normal blood donors bydensity gradient ultracentrifugation (density=1.019-1.063 g/ml) aspreviously described in Qin et al., Am. J. Physiol. 274(5 Pt 2):H1836-1840 (1998), and stored in saline-EDTA. Just prior to use, EDTAwas removed by dialysis against PBS, and the concentration of LDL wasdetermined by Lowry assay (in milligrams of LDL-associated protein).

Assessment of lipid oxidation

Stock solutions of 10 mM CuSO₄ were freshly prepared daily. Reactionmixtures containing 1 mg/mi surfactant lipids or 150 μg/ml LDL wereincubated with 10 μM CuSO₄ for 24 hours or 4 hours, respectively, in thepresence of putative oxidation inhibitors or controls at 37° C. in ashaking water bath in accordance with the method of Gelvan et al.,Biochim. Biophys. Acta 1035(3):353-360 (1990). Control reactions whichincluded LDL only, 10 μM CuSO₄ or protein controls of bovine serumalbumin (BSA), rat IgG, recombinant MBP, rat serum and human complementC1q were performed concurrently with experimental samples.Thiobarbituric acid-reactive substances (TBARS) were measured using amethod adapted from Gelvan et al., Biochim. Biophys. Acta1035(3):353-360 (1990). Samples and 0-10 μM1,1,3,3-methylmalondialdehyde standards were developed by addition of asolution composed of 0.375% thiobarbituric acid, 15% trichloroaceticacid, and 0.25N HCl at a volume ratio of 1:2, sample: developer.Following incubation at 95° C. for 30 minutes and centrifugation at14,000 rpm for 15 minutes, an aliquot was read at 540 nM in aspectrophotometer. For continuous assessment of lipid oxidation, theaccumulation of conjugated dienes during LDL oxidation was monitoredspectrophotometrically in accordance with the method of Esterbauer etal., Free Rad. Med. Biol. 13, 341-390 (1992). Mixtures of 150 μg/ml LDL,10 μM copper, and various amounts of surfactant proteins or controlproteins were placed in quartz cuvettes and allowed to oxidize at roomtemperature over 335 minutes. Conjugated diene formation was assessed bymeasuring absorbance at 234 nm in a spectrophotometer.

Detection of Protein Oxidation by Western Blot Analysis

Proteins were analyzed for carbonyl modification of amino acid sidegroups that occur during lipid oxidation, as described in Levine et al.,Methods Enzymol. 186, 464-478 (1990). Briefly, proteins that wereincluded in reactions mixtures with LDL and 10 μM copper werederivatized to 2,4-dinitrophenylhydrazones (DNP-hydrazones) by reactionwith 2,4-dinitrophenylhydrazine (DNPH), size fractionated by 8-16%SDS-PAGE under reducing conditions and electrophorectically transferredto nitrocellulose membranes (Oxyblot, Intergen). The membrane wassequentially incubated with a rabbit anti-DNP IgG and a horseradishperoxidase-conjugated goat anti-rabbit IgG. Blots were developed byHRP-dependent oxidation of a chemiluminescent substrate, and visualizedusing autoradiography.

Cell oxidation experiments

A murine macrophage cell line (RAW 264.7) was adhered to 24-well plates(2×10⁴ cells/well) in Ham's F12 media containing 10% FBS overnight at37° C. in a 10% CO₂ atmosphere. After washing, the cells were exposed to40 μM tert-butyl hydroperoxide (t-BOOH) in serum free Ham's F12 for 24hrs in the presence of various concentrations of the surfactantproteins. Viability was assessed for exclusion of the vital dye TrypanBlue.

Results

Effects of pulmonary collectins on lipid oxidation

The ability of SP-A and SP-D to inhibit the copper-induced oxidation ofa mixture of saturated and unsaturated lipids was assessed by measuringthe accumulation of thiobarbituric reactive substances (TBARS) during a24 hr incubation at 37° C., as illustrated by FIG. 1 a. Panel a setsforth data obtained when surfactant lipids were incubated with 10 μMCu²⁺ in the presence of the indicated concentrations of surfactantproteins SP-A or SP-D, while panel b sets forth data obtained when LDLparticles were incubated with 10 μM Cu²⁺ in the presence of theindicated concentrations of surfactant proteins SP-A or SP-D.

Native SP-A isolated from rat lungs inhibited oxidation of surfactantlipids in a dose-dependent fashion that was half maximal at aconcentration of 5.07 μg/ml (I.C.₅₀=8.4 nM, assuming rat SP-AM.W.=600,000 kDa) and complete at 10 μg/ml. Native SP-D also inhibitedcopper-induced surfactant lipids oxidation in a dose-dependent mannerwith maximal protection observed at doses equal to or greater than 0.5ug/ml. The I.C.₅₀ for protection by SP-D was 0.11 μg/ml (I.C.₅₀=0.2 nM,assuming rat SP-D M.W.=500,000), or approximately 35-fold lower thanSP-A, and 100-fold lower than the antioxidant serum lipoprotein ApoAIV(I.C.₅₀=50 nM). The inhibitory concentrations for both lung proteinswere well below their physiologic ELF levels, estimated to be 300-1800μg/ml for SP-A and 36-216 μg/ml for SP-D. Alkylation of SP-A and SP-Dwith iodoacetamide completely blocked the antioxidant effects of theproteins but neither the presaturation of SP-A with 70 uM copper, asindicated in Table 1, below, or the addition of 2 mM Ca²⁺ (not shown)had a significant effect on the activity. There was no protection fromoxidation by control proteins albumin, rat serum or the structurallysimilar molecule, C1q (the first component of complement), atconcentrations of 50 μg/ml, as indicated in Table 1, below. A rat serumconcentration of 500 μg/ml was required to achieve the same level ofinhibition of oxidation that occurred with about 5.0 μg/ml SP-A or 0.1-0.2 μg/ml SP-D. These data indicate that the two hydrophilicsurfactant proteins, SP-A and SP-D, protect surfactant lipids fromcopper-induced oxidation at physiologically relevant concentrations.

TABLE 1 TBARS from copper-induced oxidation of surfactant lipids or LDLSurfactant lipids LDL Malondialdehyde Malondialdehyde Reaction mixture(μM) (μM) Lipid* only 0.35 ± 0.03 0.01 ± 0.01 Lipid + Cu²⁺ 1.21 ± 0.104.15 ± 0.08 Lipid + Cu²⁺ + 10 μg/ml SP-A 0.23 ± 0.01 0.0 ± 0.0 Lipid +Cu²⁺ + 10 μg/ml Cu²⁺ 0.13 ± 0.03 ND presat SP-A Lipid + Cu²⁺ + 1 μg/mlSP-D 0.27 ± 0.07 0.0 ± 0.0 Lipid + Cu²⁺ + 20 μg/ml rMBP ND 4.47 ± 0.02Lipid + Cu²⁺ + 50 μg/ml BSA 1.14 ± 0.11 3.88 ± 0.22 Lipid + Cu²⁺ + 50μg/ml rat serum 1.69 ± 0.15 4.06 ± 0.08 Lipid + Cu²⁺ + 500 μg/ml rat0.509 ± 0.06  3.03 ± 0.96 serum Lipid + Cu²⁺ + 50 μg/ml Clq 1.15 ± 0.074.16 ± 0.05 Lipid + Cu²⁺ + 50 μg/ml IgG ND 4.15 ± 0.21 Lipid + Cu²⁺ + 10μg/ml alkylated 1.36 ± 0.18 ND SP-A Lipid + Cu²⁺ + 50 μg/ml alkylated ND4.21 ± 0.25 SP-A Lipid + Cu²⁺ + 10 μg/ml alkylated ND 3.92 ± 0.14 SP-DLipid + Cu²⁺ + 20 μg/ml ΔN1-P80, 0.21 ± 0.18 0.01 ± 0.01 N187S *Lipidused was surfactant lipids of LDL, as noted in adjacent columns. Data ismean ± S.E.M., n = 3. ND = not done Clq = the first component ofcomplement MBP = mannose binding protein ΔN1-P80, N187S = anonglycosylated neck and CLD construct containing an Asn187Ser mutation

Both SP-A and SP-D exhibited very similar antioxidant activity when thelipid components of LDL particles including unsaturated phospholipids,triglycerides, cholesterol and cholesterol esters were used as thesubstrates for lipid oxidation, as illustrated in FIG. 1 b. The I.C.₅₀for inhibition of copper-induced oxidation of LDL for SP-A and SP-D were4.75 μg/ml (7.9 nM) and 0.14 μg/ml (0.3 nM) respectively, and completeinhibition of oxidation occurred at 10 μg/ml and 1 μg/ml, respectively.In contrast, 20 μg/ml of the highly homologous collectin, recombinantrat mannose binding protein A (MBP-A) did not inhibit LDL oxidation, asindicated in Table 1, below. Rat serum, albumin, C1q and IgG had also noeffect on LDL lipid oxidation at concentrations of 50 μg/ml, asindicated in Table 1, below. Only 2.5 μg/ml SP-A or 0.05 μg/ml SP-D wererequired to provide the same level of antioxidant protection as 500μg/ml of rat serum. In the absence of oxidation inhibitors, the absoluteTBARS level following a 4 hour incubation with 10 μM copper was over 3times greater for LDL than for surfactant lipids, as indicated byTable 1. For this reason, LDL was used as the lipid substrate forkinetic experiments and mutagenesis studies of the collectin antioxidantactivities.

Kinetic analysis of c Ilectin antioxidant activity

The temporal relationship between the addition of SP-A or SP-D and theinhibition of LDL oxidation was determined spectrophotometrically bycontinuously monitoring conjugated diene accumulation associated withexposure to copper, in accordance with the method of Esterbauer et al.,Free Rad. Med. Biol. 13:341-390 (1992). The assay is based on theoxidation dependent rearrangement of 1,4 pentadienyl double bonds of LDLlipids to 1,3-butadienyl double bonds, which absorb in the ultravioletrange. In the presence of 10 μM copper at room temperature, LDLparticles resist oxidation for up to 100 minutes as endogenousantioxidants such as atocopherol are consumed. At that point, the rateof oxidation increases in proportion to the concentration of initiatingradicals, reaching a plateau when all unsaturated fatty acids areconsumed.

The data are set forth graphically in FIG. 2. According to FIG. 2,conjugated diene formation during copper-induced LDL oxidation wasmonitored spectrophotometrically by measuring absorbance at 234 nm. Inthe absence of copper (open square), only minimal LDL oxidationoccurred. Addition of 10 μM copper and (panel a) SP-A at 0 (opentriangle), 1.0 (open circle), 2.5 (closed square), 5.0 (closed circle),7.5 (closed triangle) and 10 μg/ml (cross) or (panel c) SP-D at 0 (opentriangle), 0.01 (open circle), 0.05 (closed square), 0.075 (closedcircle), 0.1 (closed triangle), 0.5 (x) and 1 μg/ml (cross) at zero timecaused a dose-dependent decrease in the slope of propagation curve, withcomplete inhibition of oxidation at 10 μg/ml SP-A or 0.5 μg/ml SP-D. Theeffect of collectin addition during the propagation phase of oxidationwas also assessed. The addition (arrows) of (panel b) SP-A at 0 μg/ml(open triangle) or 10 μg/ml SP-A at 95 (open circle), 175 (closedsquare) or 195 (closed triangle) minutes or (panel d) 0 μg/ml SP-D (opentriangle) or 1 μg/ml SP-D at 95 (open circle), 125 (closed square), or155 (closed triangle) minutes arrested lipid oxidation almostimmediately.

When SP-A (FIG. 2 a) or SP-D (FIG. 2 c) were included at zero time, theyblocked the accumulation of conjugated dienes in a dose-dependentmanner. With increasing surfactant protein concentrations, thepredominant change was a decrease in the slope during the rapidoxidation phase, consistent with inhibition of free radical chaininitiation or with free radical chain termination. The concentrations ofSP-A and SP-D that prevented conjugated diene formation were verysimilar to those that were required to block TBARS formation. When fullysuppressive concentrations of SP-A (10 μg/ml)(FIG. 2 b) or SP-D (1μg/ml) (FIG. 2 c) were added during the propagation phase of oxidation,conjugated diene formation was completely arrested at the point ofaddition. These data indicate that SP-A and SP-D directly interfere withlipid oxidation.

Pulmonary collectin domains and mechanisms in prevention of lipidoxidation

To determine the domain(s) of SP-A that are responsible for protectionfrom copper-induced oxidation of LDL and synthetic lipids, theactivities of mutant recombinant SP-As containing deletions inN-terminal domains were tested in the TBARS assay. The data are setforth graphically in FIG. 3. Various concentrations of recombinant wildtype SP-A (circle), mutant SP-A containing a deletion of thecollagen-like domain (square) and mutant SP-A containing only the neckand C-lectin domain (CLD) (triangle) were incubated with LDL in thepresence of 10 μM Cu²⁺, and lipid peroxidation was quantified by TBARSassay. Data are means, n=3-4.

Wild type recombinant SP-A inhibited copper-induced oxidation of LDL tothe half-maximal point at 2.1 μg/ml and to basal levels at 5.0 μg/ml. Amutant SP-A containing a deletion of the collagen-like region (ΔG8-P80,McCormack et al., J. Biol. Chem. 272:27971-27979 (1997)), but retainingthe N-terminal segment and interchain disulfide bounds, was nearly asactive as the wild type recombinant protein (I.C.₅₀=2.8 μg/ml). Anon-disulfide crosslinked trimeric construct composed solely of the neckand CLD region of the protein (ΔN1-P80, McCormack et al., J. Biol. Chem.274:3173-3183 (1999)) was also a potent antioxidant, with an I.C.₅₀ of6.7 μg/ml. Although carbohydrates are known to have antioxidantproperties, the protection of lipids by SP-A was not attributable to theoligosaccharide attached to Asn187, since a nonglycosylated neck and CLDconstruct containing an Asn187Ser mutation also inhibited lipidoxidation (ΔN1-P80,N187S), as indicated in Table 1. While not beingbound by theory, it is believed that the antioxidant activity of SP-A islocalized to the polypeptide sequences in the C-terminal (neck+CLD)domains of the proteins.

To determine if the surfactant proteins act as sinks for reactive lipidintermediates, covalent modification of SP-A and SP-D during LDLoxidation was assessed using a western analysis technique that detectscarbonyl adducts (Levine et al., Methods Enzymol. 186: 464-78 (1990)).As depicted by FIG. 4, oxidative modification of proteins that occurredduring copper induced LDL oxidation were determined by Western analysisusing an antibody to DNP-derivatized carbonyl moieties. Reactionmixtures contained LDL only, LDL+Cu²⁺, LDL+Cu²⁺+albumin+10 μg/ml SP-A,LDL+Cu²⁺+albumin+1 μg/ml SP-D, LDL+Cu²⁺+albumin, LDL+Cu²⁺+albumin+50μg/ml iodoacetamide treated SP-A, LDL+Cu²⁺+albumin+10 μg/mliodoacetamide treated SP-D, 50 μg/ml albumin+50 μg/ml iodoacetamidetreated SP-A, albumin+10 μg/ml iodoacetamide treated SP-D.

Oxidation of LDL produced a dense high molecular weight band thatcorresponded to B-100, the 514 kDa protein component of LDL, and severalsmaller bands. Incubation with 10 μg/ml SP-A or 1 μg/ml of SP-D blockedLDL oxidation without the appearance of carbonyl-modified species at theexpected molecular weights for SP-A or SP-D of 26-38 kDa or 42 kDa,respectively. However, when the surfactant proteins were inactivated bytreatment with iodoacetamide, neither alkylated SP-A at 50 μg/ml oralkylated SP-D at 10 μg/ml inhibited lipid oxidation. The appearance ofcarbonyl derivatized SP-A species at 26, 32 and 38 kDa in the oxidizedbut not in the minus copper control lane is consistent with oxidativemodification of the iodoacetamide-treated SP-A. Carbonyl modification ofiodoacetamide treated SP-D was more difficult to assess due to lowerlevels of SP-D in the mixture and the complexity of bands near 40 kDa.While not being bound by theory, it is believed that SP-A and SP-D blockcopper-induced lipid oxidation by a mechanism that does not includecarbonyl derivatization of the collectins themselves.

Effects of pulmonary collectins on oxidant-induced cell death

Exposure of cultured mammalian cells to tert-butylhydroperoxide (t-BOOH)promotes a variety of toxic events including depletion of glutathione,mitochondrial dysfunction and peroxidation of membrane lipids, asdescribed in Sestili et al., FEBS Lett. 457(1):139-143 (1999). Todetermine if SP-A and SP-D protect cells from oxidative stress, RAW264.7 murine macrophages were exposed to 40 μM t-BOOH for 24 hrs in thepresence of SP-A or SP-D at concentrations from 0.01 to 50 μg/ml. Celldeath was assessed by staining cells with the vital dye Trypan Blue. Asdepicted by FIG. 5, RAW 264.7 macrophages were incubated with variousconcentrations of SP-A or SP-D and 40 μM t-BOOH. Cell viability wasassessed by counting the percentage of cells that excluded the vital dyeTrypan Blue. Data are mean±S.E.M., n=3 for collectins except 50 μg/mlpoint where n=2.

It was found that both SP-A and SP-D protected RAW cells fromt-BOOH-induced death in a dose-dependent fashion that was half maximalat concentrations of 0.52 μg/ml for SP-A and 0.56 μg/ml for SP-D, andwhich reached a plateau at a concentration of approximately 1 μg/ml forboth proteins. These data indicate that SP-A and SP-D protectmacrophages from oxidant stress.

The experimental results indicate that SP-A and SP-D have potent, directantioxidant properties at concentrations that are well within theirphysiologic ranges. The antioxidant activity of SP-A is found in theC-terminal region of the protein, which includes the C-type lectindomain. The C-type lectin family is noted for functional diversity aspattern recognition dependent opsonins, cell adhesion molecules, cellsurface receptors and anti-freeze proteins (Drickamer, K. (1999) Curr.Opin. Struct. Biol. 9(5):585-590 (1999)). The antioxidant function maybe unique to the pulmonary collectin subgroup of the C-type lectins,however, since neither the structurally-related complement protein, C1q,or the highly homologous serum collectin, mannose binding protein A(MBP-A), had antioxidant activity.

The mechanism by which pulmonary collectins protect lipids fromoxidation was examined using several approaches. Collectin mediatedcopper chelation does not account for the antioxidant properties, sincethere was a greater than 10⁴ fold molar excess of copper to collectin inreactions that demonstrated complete inhibition of oxidation. Inaddition, neither the occupation of the SP-A metal binding site bycoincubation with 2 mM Ca²⁺ or presaturation of SP-A with copper blockedthe antioxidant activity of the protein. The stoichiometry of thereaction also indicates substrate sequestration is an implausibleantioxidant mechanism, since at inhibitory concentrations the molarratios of surfactant phospholipid:protein were >10⁴ for SP-A and >10⁶for SP-D. That the surfactant proteins may have altered theaccessibility of the lipid vesicles to copper or free radicals bycausing aggregation was considered, but SP-D does not aggregatephosphatidylcholine vesicles and experiments were done undercalcium-free and physiologic pH conditions which do not supportaggregation by SP-A (Efrati et al., Biochemistry 26:7986-7993 (1987) andHawgood et al., Biochemistry 24:184-190 (1985)). The proteins do notthemselves become modified during lipid oxidation and therefore do notprotect lipids by functioning as ‘suicide’ sinks for covalent attack byreactive lipid intermediates. SP-A and SP-D arrest LDL oxidation almostinstantaneously despite the inaccessibility of the hydrophilicsurfactant proteins to oxidizable substrates in the interior of theparticle. Collectively, the data indicate that the collectins directlyinterfere with lipid oxidation by inhibiting the formation of lipidradicals or acting as free radical chain terminators. The CLD of SP-Aand SP-D are rich in aromatic amino acids which are candidates for thequenching of free radicals.

The pulmonary collectins also protect growing cells from oxidant stress.The mechanism of t-BOOH-induced cell death is thought to be dependent onmetal ions (Miyata et al., Nat. Genet. 14(1), 55-61(1996)), mediated byfree radicals generated through the iron-dependent Fenton reaction(Buettner et al., Arch. Biochem. Biophys. 300(2), 535-543 (1993)) or bycopper-induced oxidation of lipid hydroperoxides (Patel et al., Biochem.J. 322(Pt 2):425-433(1997)). SP-A and SP-D are large hydrophilicmolecules that almost certainly exert their antioxidant effects in theextracellular compartment. Based on the results of the lipid oxidationexperiments, it is believed that the lung collectins protect cells byinterfering with the formation of free radicals or free radical chaintermination. It is possible, however, that the proteins alter thecellular response to oxidant stress by binding to cell surface moleculesand activating signaling pathways.

The lung is exposed to oxidant stress through inhalation of oxygenpresent in the atmosphere, the presence of ozone and trace metals in airpollutants, and oxidant species released from macrophages andneutrophils in the ELF. In addition, the ELF from normal subjects hasbeen shown to contain chelatable redox active iron that is derived fromendogenous sources (Gutteridge et al., Biochem. Biophys. Res. Commun.220(3), 1024-1027 (1996)). Although surfactant is inherently resistantto oxidation due to the preponderance of saturated phospholipids,oxidizable cholesterol and unsaturated phospholipids represent 40% ofthe weight of surfactant, and 15% of all surfactant phospholipidscontain two or more double bonds (Postle et al., Am. J. Respir. Cell.Mol. Biol. 20(1), 90-98 (1999)). The properties of SP-A and SP-D toinhibit the propagation of free radical chain reactions may preventwaves of lipid oxidation from spreading through the 100 m² surfactantinterface. Since SP-A is intimately associated with surfactant lipidsand aggregates in the airspace, and SP-D is found primarily free in theELF, while not being bound by theory, it is believed that the pulmonarycollectins perform complementary functions to protect the lipidinterfaces and the aqueous compartment of the ELF from oxidative stress.Recent data from collectin-deficient animal models is considered to beconsistent with this notion. The SP-D deficient gene-targeted mouse,which also had reduced SP-A levels, had enhanced oxidant production inthe lung and developed a surfactant lipid clearance defect (Wert et al.,Proc. Natl. Acad. Sci. USA 97(11):5972-5977 (2000); Botas et al., S.Proc. Natl. Acad. Sci. USA 95(20):11869-11874 (1998); and Korfhagen etal., J Biol Chem 273(43):28438-28443 (1998)). The lack of SP-D increasedhydrogen peroxide production by isolated alveolar macrophages, but andirect, acellular antioxidant role for SP-D was not examined in thosestudies (Wert et al., Proc. Natl. Acad. Sci. USA 97(11):5972-5977(2000)). On the other hand, the SP-A deficient gene-targeted mouse,which had normal SP-D levels, did not have a clear surfactanthomeostasis phenotype (Korfhagen et al., Proc. Natl. Acad. Sci. 93,9594-9599 (1996)). While not being bound by theory, it is believed thatcombined SP-A and SP-D deficiency in the SP-D null mouse results insurfactant oxidation that overwhelms the clearance capacity ofsurfactant metabolizing enzymes, but that sufficient SP-D is present inthe SP-A null mouse to protect the surfactant system from oxidativeinjury.

The recent observation that all of the major vertebrate groups have lungcollectins in their airspaces, including the most primitive amphibiousfish, underscores the fundamental importance of these proteins topulmonary function (Gutteridge et al., Biochem. Biophys. Res. Commun.220(3), 1024-1027 (1996) and Sullivan et al., J. Mol. Evol. 46, 131-138(1998)). The harmful effects of air breathing on the oxidation sensitivecellular and molecular components at the environmental interface of thelung may be mitigated by the presence of surfactant proteins A and D.

Purification and modification of surfactant proteins-antimicrobialproperties

Native SP-A and SP-D were isolated from the alveolar wash of rats whichhad been pretreated with intratracheal silica to enhance the collectinyield, in accordance with the method of Dethloff et al., Biochem. J.233:111-118 (1986). After centrifugation, rat SP-D was purified bymaltose-Sepharose affinity chromatography of the supernatant and ratSP-A was purified from the pellet by NaBr flotation, butanol extractionand mannose-Sepharose affinity chromatography, in accordance with themethod of McCormack et al., J. Biol. Chem. 272:27971-27979 (1997). MouseSP-D was used interchangeably with rat SP-D. All proteins wereextensively dialyzed to remove residual EDTA. For some experiments, ratSP-A and rat SP-D were alkylated by incubation with 0.5 M iodoacetamideat 37° C. in the dark for 1 hr and then extensively dialyzed. The wildtype and mutant recombinant SP-A, SP-D and MBP used in this study weresynthesized using baculovirus vectors and purified bycarbohydrate-Sepharose affinity chromatography, as previously describedin McCormack et al., J. Biol Chem. 272:27971-27979 (1997); McCormack etal., Biochemistry 36:13963-13971 (1997); and McCormack et al., J. Biol.Chem. 274:3173-3183 (1999).

SP-A and SP-D attenuate light scattering by growing E. coli in aLPS-reversible manner

Experiments were designed to assess the effect of SP-A on the growth andviability of bacterial organisms. For these experiments, SP-A isolatedfrom patients with alveolar proteinosis (APP-SP-A) was used, because itis available in greater abundance than the rat reagents. E. coli (FIGS.7A,B,D) or GBS (FIG. 7C) organisms that had been grown to stationaryphase overnight were diluted in Luria broth or Todd-Hewitt broth,respectively and agitated at 37° C. for five hours in the presence orabsence of APP-SP-A(FIGS. 7A,B,C) or SP-D (FIG. 7D). Bacterial growthwas monitored by measuring light scattering in a spectrophotometer at awavelength of 400 nm. In the absence of collecting, both GBS (FIG. 7C)and E. coli (FIGS. 7A,B,D) grew logarithmically and light scatteringincreased steadily over the six hour incubation approaching a peak 6 hr.optical density (O.D._(400 nm)) of approximately 2.0-3.0 units. APP-SP-Ahad no effect on the increase in light scattering due to growing GBS(FIG. 7C). For E. coli, however, the increase in light scattering waspartially inhibited by 25 μg/ml APP-SP-A (peak O.D._(400 nm) of 1.25units), and almost completely inhibited by 250 μg/ml APP-SP-A (maximalO.D._(400 nm) of 0.25 units) (FIG. 7A).

To determine if growth inhibition by SP-A was dependent on theinteraction of the protein with LPS on the bacterial surface, E. coligrowth inhibition was accessed by APP-SP-A in the presence and absenceof excess J5 LPS vesicles. As shown in FIG. 7 B and D, the timedependent increase in light scattering was inhibited by 100 μg/mlAPP-SP-A or SP-D and was not affected by J5 LPS vesicles alone. However,SP-A and SP-D mediated attenuation of light scattering produced bygrowing E. coli was completely and partially blocked by 300 μg/ml J5 LPSvesicles, respectively. These data indicate that SP-A and SP-D associatewith gram negative bacteria in an LPS dependent manner to reduce lightscattering from microbial proliferation.

SP-A and SP-D directly inhibit bacterial growth

The attenuation of light scattering by SP-A and SP-D could be due tobacterial aggregation, inhibition of growth or a combination of both.For SP-A, the number of CFUs at each time point over a six hourincubation decreased by 90% at 1 and 2 hrs, and then resumed alogarithmic growth rate over the next 3-5 hours. Light microscopicexamination of the bacterial cultures after 1 hour incubation withAPP-SP-A revealed extensive bacterial aggregation, which confounds theassessment of bacterial viability. A radial diffusion method wastherefore used to examine the effect of the collectins and theirindividual structural domains on the growth of E. coli that wereimmobilized in agar (FIGS. 8,9). Wells were bored into plates containingagar impregnated with 2×10⁶ E. coli/ml or GBS. Proteins to be testedwere introduced into the wells and the plates were incubated overnightat 37° C. Positive control protein lysozyme at 0.5 and 5.0 μg addedproduced dose-dependent clearing in both the E. coli plates and the GBSplates, but negative control protein albumin at 5 μg had no effect. RatSP-A and SP-D inhibited the growth of E. coli K12 (FIG. 8,9) but not GBS(not shown). The ΔN1-P80 (see mutant in FIG. 6C) SP-A also produceddetectable clearing in the E. coli plates at 0.5 μg and easily visibleinhibition of bacterial growth at 5 μg (FIG. 9). These data indicatethat SP-A inhibits the growth of E. coli (but not GBS) by a C-terminaldomain dependent mechanism. Inhibition of E. coli growth by recombinantmouse SP-A and SP-D were blocked with anti-mouse SP-A and anti-mouseSP-D antibodies, respectively (FIG. 8B) and by excess LPS vesicles (forSP-A>SP-D) (FIG. 8C). These results suggest that growth inhibition byour SP-A and SP-D preparations are LPS dependent, and that thecollectins themselves rather than copurifying antimicrobials (such asEDTA or azide that may inadvertently leach from the columns) areresponsible for the growth inhibitory effect. SP-A also inhibited thegrowth of P. aeruginosa (PAO1 mutant) in a radial diffusion assay.

Outer membrane protein A protects E. coli from growth inhibition by SP-Aand SP-D

SP-A has been reported to bind to the outer membrane protein (OmpA) ofHemophilus influenza, a porin that facilitates the uptake of nutrientsfrom the environment. The hypothesis that binding of collectins to OmpAmediates bacterial growth inhibition by SP-A and SP-D was tested. Theseexperiments were performed using a genetically engineered clinicalisolate of E. coli that was rendered OmpA deficient (ΔOmpA) bytransposon mutagenesis. SP-A and SP-D inhibited the growth of theparental E. coli strain (E69), but growth inhibition was increased(judged by the radius of clearing around the well) by the geneticdeletion of OmpA (FIG. 10). Replacement of OmpA in the ΔOmpA bacteria byplasmid driven OmpA expression attenuated growth inhibition by SP-A andSP-D, but the ΔOmpA organism that contained an empty vector control wasinhibited to the same extent as the ΔOmpA organism. These data indicatethat OmpA protects E. coli from growth inhibition by SP-A and SP-D andsuggests that SP-A and SP-D may exert their effects at the level of thecell membrane.

SP-A inhibits protein synthesis in Histoplasma capsulatum

As depicted by FIG. 11, SP-A directly inhibits the incorporation of³H-leucine into H. capsulatum in a dose-dependent manner that reaches amaximal effect at about 150 μg/ml APP-SP-A. These data indicate that theinhibition of microbial growth by the collectins is not specific tobacteria.

SP-A can inhibit and reverse light scattering due to aggregation ofvesicles composed of E. coli Phospholipids

As depicted by FIG. 12, E coli phospholipid vesicles generated by probesonication were aggregated by addition of Ca²⁺(□). SP-A decreasedaggregation when added at 0 time (Δ), and SP-A added at 8 minutesreversed established aggregation (μ) in a manner that was similar toaddition of 10 mM EDTA at 8 minutes (□). SP-A also inhibited theaggregation of E. coli J5 LPS, but did not reverse establishedaggregation by J5 LPS vesicles (not shown). In contrast, SP-A mediatesaggregation of surfactant liposomes. This system will be useful formodeling the structural basis of the interaction between collectins andbacterial membranes.

SP-A and SP-D form complexes with metals.

SP-A and SP-D block lipid peroxidation (LPO) that is produced by 24 hrincubation by 10 μM copper or iron. Recent preliminary data presentedabove indicates that the antioxidant properties of SP-A and SP-D requirecomplex formation with transition metals (FIG. 13). In theseexperiments, LPO of model surfactant lipids was initiated with the freeradical generating compound 2,2′-azobisisobutyronitrile (AIBN). SP-A andSP-D did not inhibit LPO, unless copper was added at low concentrations.Copper did not contribute to LPO under the experimental conditionsemployed, due to the short incubation period (3 hours). Zinc could notsubstitute for copper in these experiments, but iron could (not shown).These data indicate that SP-A and SP-D form complexes with transitionmetals. The association of the collectins with metals may not onlyconfer antioxidant properties on the proteins but may also destabilizebacterial membranes or limit bacterial growth by producing an iron (orother metal) deficient environment.

Through-out the specification, parts and percentages are by weightunless otherwise indicated. Additional embodiments and modificationswithin the scope of the claimed invention will be apparent to one ofordinary skill in the art. Accordingly, the scope of the presentinvention shall be considered in the terms of the following claims, andis understood not to be limited to the details, examples or the methodsdescribed in the specification.

1. A method of treating a patient for acute lung injury comprisingadministering to the patient an effective antimicrobial amount of anantimicrobial lung surfactant protein compound, wherein theantimicrobial lung surfactant protein compound is a peptide comprisingthe amino acid sequence:Met-Phe-Leu-Lys-Ala-Val-Val-Leu-Thr-Val-Ala-Leu-Val-Ala-Ile-Thr-Gly-Thr-Gln-Ala-Glu-Val-Thr-Ser-Asp-Gln-Val-Ala-Asn-Val(SEQ ID NO:1).
 2. A method of treating patient for acute lung injurycomprising administering to the patient an effective antioxidationamount of an antioxidant lung surfactant protein compound comprising theamino acid sequence:Met-Phe-Leu-Lys-Ala-Val-Val-Leu-Thr-Val-Ala-Leu-Val-Ala-Ile-Thr-Gly-Thr-Gln-Ala-Glu-Val-Thr-Ser-Asp-Gln-Val-Ala-Asn-Val(SEQ ID NO:1).